Delivery construct for antisense nucleic acids and methods of use

ABSTRACT

A novel nucleic acid construct for delivery of antisense targeting sequences is provided. The construct includes intact stem loop structures and an antisense nucleic acid. Optionally, a ribozyme nucleic acid is included in the construct. The construct is useful for inhibition of selected genes in a cell. This allele-specific targeting is also useful in combination with replacement gene therapy.

FIELD OF THE INVENTION

This invention relates generally to the field of delivery vehicles fornucleic acid molecules and specifically to a novel construct fordelivery of antisense targeting sequences to a cell.

BACKGROUND OF THE INVENTION

The use of antisense oligonucleotides offers advantages over othertherapeutic regimes due to their potential for target specificity. Forexample, conventional chemotherapy for neoplastic and virus-relateddiseases has the disadvantage of systemic toxicity. The therapeuticindex for chemotherapeutic agents is relatively narrow, since suchagents are unable to distinguish between normal and diseased cells.Antisense oligonucleotides have the potential to be many orders ofmagnitude more specific than traditional drugs due to their greaternumber of interactions with a particular target site. In theory, anoligonucleotide of more than 15-17 nucleotides in length could have thebase pairing specificity to interact with only one target gene withinthe entire human genome. Thus, antisense oligonucleotides have thepotential specificity that could serve as a powerful tool for the studyof specific gene function and as therapeutic agents for disease-causinggenes.

In contrast to drugs, antisense molecules are relatively simple todesign. The interaction between an antisense oligonucleotide and atarget mRNA is governed primarily by the sequence of the target.Oligonucleotides targeting the start codon and extending upstream ordownstream have been shown to be effective. Similarly, oligonucleotidesthat are complementary to the splice sites have proved effective.

Antisense technologies for the targeted inhibition of gene expressioncould provide an effective strategy for the management of inheriteddisorders with dominant-negative or gain-of-function pathogeneticmechanisms, for the suppression of oncogenes, or for the control of avariety of infectious agents. Pathologic disorders that are currentlytargeted by antisense therapeutics include viral infections,inflammatory disorders, cardiovascular disease, cancers, geneticdisorders and autoimmune diseases. Synthetic oligodeoxynucleotides(ODNs), especially phosphorothiates and methylphosphonates, offer theadvantage of enhanced stability in biological fluids and an effectivelylimitless supply.

Antisense oligonucleotides are also useful for the production oftransgenic animals having alterations at the germline level, such asknockout mutations, which can be used for the study of new genes or thestudy of the function of a known gene. Further, antisense technologycombined with gene therapy is useful for example, for suppression ofexpression of a mutant gene product. Such gene therapy would be mostadvantageous in combination with a replacement regimen utilizing theAnormal≅ gene to provide a Anormal≅ gene product.

Unfortunately, the effective use of antisense oligonucleotides has beenlimited due to several problems. Disadvantages include the transientnature of ODNs, and their toxicity and propensity for producingnon-sequence specified biological effects. Other disadvantages includelow expression or limited stability of complementary RNAs which resultin their nonspecific targeting or low efficiency of target inhibition.Antisense oligonucleotides are often poorly taken up by cells andtherefore may never reach their target site. Often, antisenseoligonucleotides do not reach the nucleus of a cell once administered,the site of their RNA and DNA targets. In certain applications theantisense molecules are microinjected directly into the cells. Thistechnique works well in the laboratory, however, it cannot be applied topatients. Many of the studies with antisense show that gene expressionis suppressed by 80-90% of the normal level, however, such reduction isnot typically sufficient to reduce the biological effect, e.g., 10-20%expression is sufficient to maintain the biological function sought tosuppress.

There is a need to develop a delivery system for antisense moleculesthat gives the antisense enhanced stability, for example by beingresistant to nuclease activity or by being enriched in the nucleus,while still allowing specificity of the antisense for its target RNA orDNA. Such a system would provide effective targeting of the message withthe end result being significant inhibition of expression of aparticular gene.

SUMMARY OF THE INVENTION

The present invention provides a novel nucleic acid construct fordelivery of antisense targeting sequences for inhibition of selectedgenes in a cell. The construct includes intact stem loop structures andan antisense nucleic acid. This allele-specific targeting is also usefulin combination with replacement gene therapy.

In a first embodiment, the invention provides a nucleic acid constructfor suppressing gene expression. The construct includes a 5′ stem loopstructure, an antisense nucleic acid, and a 3′ stem loop structure. SuchAunmodified≅ or Aintact≅ stem loop structures flank the antisensenucleic acid so that the antisense oligonucleotides can readily interactwith any target sequence. In a preferred embodiment, the stem loopstructures are U snRNA stem loops, and most preferably, U1 snRNa stemloops. The construct provides a cloning site outside of the stem loopstructures, thus rendering the stem loop structures Aunmodified,≅ intowhich virtually any antisense oligonucleotide could be inserted. Incontrast to previous antisense delivery vehicles which have beendesigned to interfere with mRNA splicing, the construct of the presentinvention does not necessarily affect the splicing machinery so as notto disrupt normal cellular mRNA processing. In other words, whileprevious constructs retain elements that interact with spliceosomeproteins, the construct of the invention eliminates such elements.Preferably, a U1 snRNA promoter is included in the construct.Optionally, a ribozyme nucleic acid is included in the construct.

In another embodiment, the invention provides a method for suppressionof gene expression whereby a suppressive-effective amount of the nucleicacid construct of the invention is administered to a cell therebysuppressing expression of the gene. The method is particularly usefulwhen used in combination with replacement gene therapy which provides agene which encodes and can reconstitute the wild-type protein and whichis resistant to targeting by the antisense and/or ribozyme.

The seminal discovery of the delivery vehicle of the invention nowallows transgenic knockout animals to be created for the study of knownor as of yet unknown genes. For example, phenotypic consequences of lossof function due to targeted disruption of a gene(s) can be determined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustrative expression construct of the invention.

FIG. 2 shows the sequence and predicted structure of chimerictranscripts derived from pU1/FIB.

FIG. 3 shows an immunohistochemical analysis of MG63 cells that werestably transfected with an expression construct (pZeoSVLacZ, Invitrogen)lacking targeting sequence (panels A and C) or pU1/FIB (panels B and D).

FIG. 4 shows a Northern blot analysis of 3.5 μg of poly(a) RNA extractedfrom confluent MG63 cells that were untransfected (lane 1), or stablytransfected with either pZeoSVLacZ (Lane 2) or pU1/FIB (lane 3-5).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a novel nucleic acid construct whichprovides a general means of delivery for antisense targeting sequences.The construct includes stabilizing structural elements, stem loopstructures, which increase the effectiveness of the targeting construct.Also included is a method of suppression of gene expression utilizingthe novel construct of the invention.

The nucleic acid construct of the invention which is useful forsuppressing gene expression includes 5′ and 3′ stem loop structures andan antisense nucleic acid. While not wanting to be bound by a particulartheory, it is believed that the stem loop structures provide enhancedstability of the targeting molecules by conferring resistance toexonucleases and also promote duplex formation and stability.

The Astem loop≅ structures refer to nucleic acid structures that havefolding patterns which form hairpins and flank the antisense targetingsequence. The stem loop structures are preferably unmodified, naturallyoccurring structures. Alternatively, one of skill in the art would beable to synthesize such structures to Amimic≅ the naturally occurringstructures. The preferred stem loop structures in the construct of theinvention are unmodified U snRNA structures. The term Aunmodified≅ meansthat the folding pattern of the stem loop structure is not compromisedby alterations in the nucleic acid sequence of the naturally occurringmolecule. For example, it is understood that alterations which include,but are not limited to, mutations, insertions, deletions andsubstitutions of one or more nucleotides can be made within the sequenceof the stem loop, as long as the stabilization function and hairpinformation of the stem loop is maintained.

Preferably, the stem loop structures are derived from or synthesizedaccording to the nucleotide sequence of the U snRNA stem loopstructures. An exemplary construct utilizing U1 snRNA stem loops isshown in FIG. 1 herein. One can produce a chimeric construct having a 5′stem loop structure from one species and a 3′ structure from a secondspecies. For example, a 5′ stem loop is derived from human U snRNA andthe 3′ stem loop derived from a yeast U snRNA. Other snRNA stem loopstructures can be derived from other mammals (e.g., porcine, bovine), oryeast for example. Other chimeric constructs can be prepared using a 5′stem loop structure from one snRNA (e.g., U1) and a 3′ stem loopstructure from another snRNA (e.g., U3). Virtually any combination ofstem loop structures is envisioned in the construct of the invention.

As used herein, the term “nucleic acid=or nucleic acid sequence” refersto a polymer of deoxyribonucleotides or ribonucleotides, in the form ofa separate fragment or as a component of a larger construct. Forexample, nucleic acids can be assembled from cDNA fragments or fromoligonucleotides to generate a synthetic gene which is capable of beingexpressed in a recombinant transcriptional unit. Polynucleotide ornucleic acid sequences of the invention include DNA, RNA and cDNAsequences.

Nucleic acid sequences utilized in the invention can be obtained byseveral methods. Sequences for specific genes or stem loop structurescan determined from published sequences and can also be found inGenBank, National Institutes of Health computer database. Nucleic acidscan then be chemically synthesized by standard methods for example.

The construct of the invention includes an antisense nucleic acidflanked by the stem loop structures. The antisense nucleic acid can bedirected toward any target nucleic acid, and preferably to a targetmessage (mRNA). Any antisense which includes sequences capable ofhybridizing with its complementary target can be used in the constructof the invention. For example, antisense sequences can be directed tothe 5′ or 3′ termini of the target message, to splice junctions, or tointernal sequences. One of skill in the art will readily be able todetermine which sequences to use as the appropriate antisense nucleicacid construct. AAntisense nucleic acids≅ are DNA or RNA molecules thatare complementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely to betoxic than larger molecules when introduced into the target cell. Theuse of antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988). Anantisense core nucleic acid may contain about 10 nucleotidescomplementary to the target message. Examples of target messages includetranscription regulatory factors (e.g., rent-1), viral encoded proteins(e.g., human papilloma virus E6, human immunodeficiency virus tat),(e.g., haluronic acid synthase), structural proteins (e.g., fibrillin),cytokines, oncogenes and growth factors (e.g., interleukins), etc. Itshould be noted that the antisense nucleic acid can be useful forreducing the expression of either normal or aberrant genes.

The antisense nucleic acid can be used to block expression of a mutantprotein or a dominantly active gene product, such as amyloid precursorprotein that accumulates in Alzheimer=s disease. Such methods are alsouseful for the treatment of Huntington=s disease, hereditaryParkinsonism, and other diseases. Antisense nucleic acids are alsouseful for the inhibition of expression of proteins associated withtoxicity or gene products introduced into the cell, such as thoseintroduced by an infectious agent (e.g., a virus).

It may be desirable to transfer an antisense nucleic acid encoding abiological response modifier in order to reduce the expression of suchbiological response modifier. Included in this category are nucleicacids encoding immunopotentiating agents including a number of thecytokines classified as “interleukins”. These include, for example,interleukins 1 through 12. Also included in this category, although notnecessarily working according to the same mechanisms, are interferons,and in particular gamma interferon (γ-IFN), tumor necrosis factor (TNF)and granulocyte-macrophage-colony stimulating factor (GM-CSF). It may bedesirable to deliver such antisense nucleic acids to cells of the immunesystem to treat enzymatic related disorders or immune defects. Antisensenucleic acids can be used to reduce expression of growth factors, toxicpeptides, ligands, receptors, or other physiologically importantproteins.

The construct of the invention may further include a ribozyme nucleicacid. Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because the ribozymes are engineered to besequence-specific, only mRNAs with sequences complementary to theconstruct containing the ribozyme are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, et al., Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences fromabout 3 to 18 bases in length. The longer the recognition sequence, thegreater the likelihood that the sequence will occur exclusively in thetarget mRNA species. Consequently, hammerhead-type ribozymes arepreferable to tetrahymena-type ribozymes for inactivating a specificmRNA species. The preferred ribozyme of the present invention is ahammerhead type ribozyme. The target message is required to contain aribozyme cleavage site sequence such as 5′-GUC-3′ or 5′-GUA-3′ when aribozyme is included in the construct.

The construct may also optionally include a triplex oligomer. Use of anoligonucleotide to stall transcription is known as the triplex strategysince the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design,6(6):569, 1991).

The phrase “nucleic acid sequence expressing a product of interest”refers to a nucleic acid sequence which when expressed results in aproduct selected from a protein or antisense RNA, for example. The term“structural gene” excludes the non-coding regulatory sequence whichdrives transcription. The structural gene may be derived in whole or inpart from any source known to the art, including a plant, a fungus, ananimal, a bacterial genome or episome, eukaryotic, nuclear or plasmidDNA, cDNA, viral DNA or chemically synthesized DNA. A structural genemay contain one or more modifications in either the coding or theuntranslated regions which could affect the biological activity or thechemical structure of the expression product, the rate of expression orthe manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions and substitutions ofone or more nucleotides. The structural gene may constitute anuninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions.

The term “operably associated” refers to functional linkage between theregulatory (e.g., promoter) sequence and the nucleic acid regulated bythe regulatory sequence. The operably linked regulatory sequencecontrols the expression of the product expressed by the structural geneor antisense. The regulatory sequence may be homologous or heterologousto the desired gene sequence. A wide range of promoters may be utilized,including viral or mammalian promoters. Cell- or tissue-specificpromoters can be utilized to target expression of gene sequences inspecific cell populations. Suitable mammalian and viral promoters forthe present invention are available in the art. A preferred promoter inthe construct of the invention is a U snRNA promoter, and mostpreferably, a U1 snRNA promoter.

The choice of a particular heterologous promoter region as a Aregulatorynucleotide sequence≅ of the invention, is dictated by the spatial andtemporal pattern of expression that is desired for the transactivatorgene and ultimately for the target transgene. Promoter regions of theinvention include eukaryotically derived promoters which predominantlydirect expression in, for example, the reproductive system (e.g.,breast, ovary, testes); the musculoskeletal system (e.g., muscle orjoint tissue); the cardiovascular system (e.g., capillaries or heart);the respiratory system (e.g., lung or nasal passages); the urologicalsystem (e.g., kidney or bladder); the gastrointestinal system (e.g.,pancreas, liver, or intestines); the immune system (e.g., thymus,spleen, or circulating immunological cells); the endocrine system (e.g.,pituitary, gonads, and thyroid); the nervous system (e.g., neurons); andthe hematopoietic system (e.g., bone marrow and peripheral blood).Further, promoters of the invention include, but are not limited to theelastase promoter (including its enhancer; expression in pancreaticacinar cells); the alpha-A-crystallin promoter (expression in the eyelens tissue); the insulin promoter (including enhancer; expression inthe pancreatic beta cells) and the albumin promoter region, includingits enhancer.

Alternatively, non-eukaryotically-derived promoters such asvirally-derived and prokaryotically-derived promoters are also includedin the present invention. Such virally-derived promoters include, butare not limited to MMTV and MoSV LTR, SV40 early region, RSV or CMV LTR,which direct expression of viral or host genes in specific tissues andin many cell types.

Promoters useful in the invention include both constitutive andinducible natural promoters as well as engineered promoters. To be mostuseful, an inducible promoter should 1) provide low expression in theabsence of the inducer; 2) provide high expression in the presence ofthe inducer; 3) use an induction scheme that does not interfere with thenormal physiology of the cell; and 4) have no effect on the expressionof other genes. Both constitutive and inducible promoters and enhancerswill be known to those of skill in the art. The promoters used in theconstruct of the present invention may be modified, if desired, toaffect their control characteristics.

The regulatory nucleotide sequence of the invention may also includeAenhancer≅ regions. Enhancers, as used herein, refer to DNA sequenceswhich affect transcription of a gene by RNA polymerase II, withoutregard to position or orientation. An enhancer region can be thousandsof base pairs from a transcription unit and still affect itstranscription. Example of enhancers known in the art include theimmunoglobulin heavy (mu) chain or light (kappa) chain enhancers and theSV40 72-base pair repeats.

It may be desirable to deliver the construct of the invention directlyto the cell without the requirement for expression, therefore, apromoter or other regulatory nucleic acid is optional. One example of atargeted delivery system for antisense polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes or liposome formulations. The preferred colloidal system ofthis invention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. Theseformulations may have net cationic, anionic or neutral chargecharacteristics are useful characteristics with in vitro, in vivo and exvivo delivery methods. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 Φm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

The construct of the invention can also be delivered as a naked Ageneexpression vector≅. This means that the construct is not associated witha delivery vehicle (e.g., liposomes, colloidal particles and the like).One of the principal advantages touted for naked DNA vectors has beenthe lack of immune responses stimulated by the vector itself.

The backbone or framework of the construct preferably includes U1 snRNAnucleic acid sequences since U1 snRNA is enriched in the nucleus, iswidely dispersed in the nucleoplasm, and is abundantly expressed. In theexemplary construct shown in FIG. 1, an Aantisense targeting core≅ wassubstituted for the Sm protein binding site between the two naturallyoccurring U1 snRNA hairpins. This core contains a 30-35 basepairsequence that is directly complementary to the target message,interrupted in its center by an autocatalytic hammerhead ribozyme loop.Duplex formation aligns the ribozyme with the GUC or GUA consensus sitesfor ribozyme cleavage within the target message. An illustrativeexpression construct of the invention which targets RENT1 transcripts,which are involved in the NMRD pathway, described below, is shown inFIG. 1. The term rentl and RENT1 (regulator of nonsense transcripts)refers to the gene and protein, respectively, from either the murine orhuman species.

The construct described above is useful for suppressing gene expression.For example, it may be desirable to modulate the expression of a genewhen it is over-expressed. Where a cell proliferative disorder isassociated with the expression of a gene, nucleic acid sequences thatinterfere with the gene=s expression at the translational level can beused. This approach utilizes, for example, antisense nucleic acid,ribozymes, or triplex agents to block transcription or translation of aspecific mRNA, either by masking that mRNA with an antisense nucleicacid or triplex agent, or by cleaving it with a ribozyme, as describedabove.

The construct may also optionally include a 5′ cap structure, such as atrimethylguanosine cap.

In another embodiment, the present invention also includes a method forsuppression of gene expression which includes administering to a cell asuppressive-effective amount of the nucleic acid construct of theinvention so that the expression of the gene is suppressed. The termAsuppressive-effective≅ amount means that amount of the construct, andthus antisense, administered is sufficient to suppress the expression ofthe target, e.g., inhibit translation of mRNA, by at least 75% of thenormal expression, and preferably by at least 90%. The effectiveness ofthe construct can be determined phenotypically or by standard Northernblot analysis or immunohistochemically, for example. Other standardnucleic acid detection techniques or alternatively immunodiagnostictechniques will be known to those of skill in the art (e.g., Western orNorthwestern blot analysis).

The present invention also provides a method of gene therapy for thetreatment of cell proliferative or immunologic disorders and diseasessuch as which are mediated by various proteins. The term“cell-proliferative disorder” denotes malignant as well as non-malignantcell populations which often appear to differ from the surroundingtissue both morphologically and genotypically. Such disorders may beassociated, for example, with abnormal expression of a gene. “Abnormalexpression” encompasses both increased or decreased levels ofexpression, as well as expression of a mutant form of a gene such thatthe normal function of the gene product is altered. Abnormal expressionalso includes inappropriate expression of during the cell cycle or in anincorrect cell type. The antisense polynucleotide is useful in treatingmalignancies of the various organ systems. Such therapy would achieveits therapeutic effect by introduction of the antisense construct intocells having the proliferative disorder. Delivery of antisensepolynucleotide, can be achieved using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system as describedabove.

The construct may also be useful in treating malignancies of the variousorgan systems, such as, for example, lung, breast, lymphoid,gastrointestinal, and genito-urinary tract as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer, leukemia, breast cancer, non-small cellcarcinoma of the lung, cancer of the small intestine, and cancer of theesophagus.

The method is also useful in treating non-malignant orimmunologically-related cell-proliferative diseases such as psoriasis,pemphigus vulgaris, Bechet's syndrome, acute respiratory distresssyndrome (ARDS), ischemic heart disease, post-dialysis syndrome,rheumatoid arthritis, acquired immune deficiency syndrome, vasculitis,lipid histiocytosis, septic shock and inflammation in general.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, adeno-associated virus, herpes virus,vaccinia, or, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when thesubject is a human, a vector such as the gibbon ape leukemia virus(GaLV) is utilized. A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a sequence of interest intothe viral vector, along with another gene which encodes the ligand for areceptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Preferredtargeting is accomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome or attached to a viralenvelope to allow target specific delivery of the retroviral vectorcontaining the antisense polynucleotide.

Administration of the construct of the invention can be in vivo, invitro or ex vivo.

The method of the invention also envisions gene replacement therapy toreconstitute expression of a wild-type protein in cells expressing adefective protein or, in the cae of a heterozygote, a defective and aAnormal≅ protein. Therefore, in another embodiment the inventionprovides a method of suppressing gene expression as above and furthercomprises administering a modified nucleic acid encoding a wild-typepolypeptide corresponding to the gene product of the gene beingsuppressed, wherein the modified nucleic acid is resistant to ribozymecleavage and/or antisense inhibition. Due to degeneracy in the geneticcode, multiple nucleotide substitutions can be made across the antisenseoligonucleotide target region of the coding sequence within anexogenously supplied expression construct encoding the wild-type gene ofinterest. Such substitutions would inhibit duplex formation between thegene suppressed and the antisense nucleic acid and transcripts derivedfrom the replacement Anormal≅ gene, while maintaining the fidelity ofthe amino acid sequence. In other words, the replacement gene wouldprovide a protein with the correct amino acid sequence utilizing codonswith nucleotides that are not recognized by the antisense constructand/or ribozyme. In this method, it is therefore possible to Aknockout≅a defective copy of a gene/mRNA and introduce a Anornal≅ copy resistantto the Aknockout≅ construct. Thus, both normal and aberrant expressionof a gene can be blocked by introduction of the antisense construct ofthe invention; to restore normal function of the gene, a copy of thewild-type gene is introduced, that, while coding for a normal protein,is resistant to duplex formation with the antisense construct and/orribozyme cleavage.

There are a number of inherited diseases in which defective genes may bereplaced including: lysosomal storage diseases such as those involvingβ-hexosamiNidase or glucocerebrosidase; deficiencies in hypoxanthinephosphoribosyl transferase activity (the ALesch-Nyhan≅ syndrome);amyloid polyneuropathies (prealbumin); Duchenne=s muscular dystrophy,and retinoblastoma, for example.

Pathologic disorders that can be targeted using the method of theinvention include but are not limited to viral infections, inflammatorydisorders, cardiovascular disease, cancers, genetic disorders andautoimmune diseases.

The method of the present invention is also useful for suppression of adominant negative mutation. The term Adominant negative≅ is a term ofart and refers to expression of a gene resulting in a gene product thatactively interferes with the function of a Anormal-, endogenous protein.Thus, a mutant protein or dominantly active gene product, such asamyloid precursor protein that accumulates in Alzheimer=s disease can beblocked. The dominant negative phenotype is conveyed by the expressionof the mutant protein that interferes with the function of the normalprotein. Such an effect is similar to the effect of dominance of oneallele of a pair of alleles encoding homologous genes on a pair ofhomologous chromosomes such that the phenotypic effect of the one alleleexerts a deleterious controlling influence over the other allele.

The nucleic acid construct can be utilized in the discovery of unknownhuman genes, for example, with a priori knowledge of the gene inquestion. In one embodiment of this invention, cells are isolated froman individual displaying a particular phenotype which is suspected ofbeing caused by a gene or genes containing nonsense mutations. A subsetof these cells is contacted (e.g., by transfecting the cells), with theconstruct that contains at least an antisense nucleic acid forsuppression or inhibition of the expression or function of a gene (e.g.,Rent-1 gene or other genes involved in NMRD) or its gene product in thecells. Levels of mRNA containing nonsense mutations in these cells areelevated to higher levels. Using standard techniques of differentialdisplay of mRNA, or genetic subtraction techniques commonly applied tocDNAs, the mRNA containing nonsense mutations can be enriched and thenisolated by comparing the mRNA populations of the original cells withthe mRNA populations in cells containing the antisense.

In a further embodiment, a transgenic animal can be developed using thenovel construct and method of the invention in order to identify theimpact of increased or decreased gene expression on a particular pathwayor phenotype. Protocols useful in producing such transgenic animals aredescribed below. The protocol generally follows conventional techniquesfor introduction of expressible transgenes into mammals. Those ofordinary skill in the art will be familiar with these applications andwill be able to apply the techniques in the context of the presentinvention without undue experimentation.

For example, embryonic target cells at various developmental stages canbe used to introduce transgenes. Different methods are used depending onthe stage of development of the embryonic target cell. The zygote is thebest target for microinjection. In the mouse, the male pronucleusreaches the size of approximately 20 micrometers in diameter whichallows reproducible injection of 1-2 pl of DNA solution. The use ofzygotes as a target for gene transfer has a major advantage in that inmost cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster, et al., Proc. Natl. Acad. Sci. USA82:4438-4442, 1985). As a consequence, all cells of the transgenicnon-human animal will carry the incorporated transgene. In general, thiswill also be reflected in the efficient transmission of the transgene tooffspring of the founder since 50% of the germ cells will harbor thetransgene. Microinjection of zygotes is a preferred method forincorporating transgenes in practicing the invention.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenisch, Proc. Natl. Acad. Sci USA73:1260-1264, 1976). Efficient infection of the blastomeres is obtainedby enzymatic treatment to remove the zona pellucida (Hogan, et al.,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). The viral vector system used to introducethe transgene is typically a replication-defective retrovirus carryingthe transgene (Jahner, et al., Proc. Natl. Acad. Sci. USA, 82:6927-6931,1985; Van der Putten, et al., Proc. Natl. Acad. Sci USA 82:6148-6152).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Steward, et al., EMBO J, 6:383-388, 1987).

Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner, etal., Nature, 298:623-628, 1982). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retroviral infectionof the midgestation embryo (Jahner, et al., supra, 1982).

A third type of target cell for introduction of heterologous nucleicacid sequences is the embryonal stem cell (ES). ES cells are obtainedfrom pre-implantation embryos cultured in vitro and fused with embryos(Evans, et al., Nature, 292:154-156, 1981; Bradley, et al., Nature,309:255-258, 1984; Gossler, et al., Proc. Natl. Acad. Sci USA,83:9065-9069, 1986; and Robertson, et al., Nature, 322:445-448, 1986).Transgenes can be efficiently introduced into the ES cells by DNAtransfection or by retrovirus-mediated transduction. These transformedES cells can thereafter be combined with blastocysts from a non-humananimal. The ES cells will thereafter colonize the embryo and contributeto the germ line of the resulting chimeric animal (see for review,Jaenisch, Science, 240:1468-1474, 1988). Any ES cell may be used inaccordance with the present invention. It is, however, preferred to useprimary isolates of ES cells. Such isolates may be obtained directlyfrom embryos such as with the CCE cell line disclosed by Robertson, E.J., in Current Communications in Molecular biology, Capecchi, M. R.(Ed.) Cold Springs Harbor Press, Cold Springs Harbor, N.Y. (1989),pp.39-44), or from the clonal isolation of ES cells from the CCE cellline (Schwartzberg, P. A. et al., Science 246:799 (1989). ES cells maybe derived or isolated from any species, although cells derived orisolated from mammals such as rodents, rabbits, and non-human primatesare preferred.

Construction of transgenes can be performed by those of ordinary skillin the art using the teachings herein. One of ordinary skill in the artcan Aknock out≅ a gene in mice by targeted disruption of the gene. Thiscan be accomplished by homologous recombination in murine embryonic stem(ES) cells using standard techniques. The clinical and cellularconsequences of targeted disruption can be investigated in multiplegenetic backgrounds including inbred strains, strains with manyundefined nonsense alleles, and strains of known mutant genotype todetermine if: (a) targeted disruption of the gene can effect a loss offunction; (b) loss of function can have an phenotype consequences (i.e.,the creation of knockout phenotypes), (c) loss of the gene productunmasks the effects of protein that can be expressed from Aphysiologic≅somatically acquired or inherited nonsense alleles upon transcriptstabilization.

The cre/lox system as described in U.S. Pat. No. 4,959,317, incorporatedherein by reference, can be utilized in the production of transgenicanimals. A first and second loxP DNA sequence is introduced into cellsconnected by a pre-selected antisense or replacement gene, hereinreferred to as a Atarget transgene≅. The Atarget transgene≅ of interestcan be a complete gene or any other sequence of nucleotides includingthose of homologous, heterologous, or synthetic origin. The targettransgene sequence can be for example, an antisense or replacement genefor a structural protein, an enzyme, or a regulatory molecule. Thetarget transgene may also be a gene of undetermined function. Usingtissue-specific or developmentally-specific regulatory sequences (asdescribed above) to direct expression of the target transgene, afunction could be identified. If the first and second lox sites have thesame orientation (direct repeats), activation of the regulatorynucleotide sequence of the transactivator transgene results in adeletion of the target transgene DNA, such that ablation or modificationof activity results. If the first and second lox sites have oppositeorientation (inverted repeats), activation of the regulatory nucleotideproduces an inversion of the nucleotides sequence of the targettransgene.

The construct of the invention may be used to introduce DNA sequencesinto the germ line cells of Anon-humans≅ to create transgenic animals.The preferred animal of the invention is a mouse. However, othernon-humans of the invention include but are not limited to other rodents(e.g. rat, hamster), rabbits, chickens, sheep, goats, fish, pigs,cattle, and non-human primates.

In yet another embodiment, an antisense library can be constructed usingthe nucleic acid construct of the invention in order to identify novelgenes. For example, a U1 construct library containing antisense coresequences (e.g., random sequences or sequences derived from a cDNAlibrary) is transfected into host cells and clones exhibiting a desiredphenotype are identified. Such clones are then examined to determine thenature of the regulatory sequence in the construct. Therefore, novelgenes having specific functions can be identified. For example, cellsthat acquire a transformed phenotype may contain an antisense moleculefor a novel tumor suppressor gene. A match between a given suppressivenucleic acid and a previously cloned gene can be made using standardtechniques (e.g., BLAST search algorithm).

The following Examples are intended to illustrate, but not to limit theinvention. While such Examples are typical of those that might be used,other procedures known to those skilled in the art may alternatively beutilized.

EXAMPLES

The following examples provide a description of construction of anexemplary nucleic acid construct of the invention, utilizing 5′ and 3′unmodified stem loop structures from U1 snRNA which flank an antisensemolecule directed toward the fibrillin-1 gene (mRNA).

Example 1

An antisense expression construct was constructed that incorporatesseveral potentially enhancing features. The pU1/FIB vector wasconstructed on the backbone of the pZeoSV (Invitrogen)prokaryotic/eukaryotic expression vector. The SV40 promoter,polyadenylation site and polylinker were excised from pZeoSV at theBamHI sites. A U1 snRNA expression cassette cloned into pUC13 wasexcised with BamHI digestion and ligated into the BamHI sites of themodified pZeoSV. Two rounds of site-directed mutagenesis (Deng, et al.,Anal. Biochem., 200, 81; 1992) were then performed to change fournucleotides flanking the Sm protein restriction sites (pZeoU1EcoSpe).Complementary oligonucleotides that encode the antisense>core=sequence,shown in FIG. 1, including the 24 highly conserved nucleotides ofhammerhead ribozymes (Cech, et al., Ann. Rev. Biochem., 55:599, weresynthesized and annealed at 40E C such that the remaining 5′ and 3′overhangs were exactly complementary to the overhangs left by EcORI andSpeI digestion. The sequences of the oligonucleotides were as follows:

-   5′-AATTGGCGATCTCCAGCACTGATGAGTCCGTGAGG ACGAAACGCCCTCGACGCAT-3′ (SEQ    ID NO:1),-   5′-CTAGATGCGTCGAGGGCGTTTCGTCCTCACGGACTC ATCAGTGCTGGAGATCGCC-3′ (SEQ    ID NO:2) (sense and antisense, respectively). The resulting duplex    was ligated into the EcORI and SpeI sites of pZeoU1EcoSpe to create    pU1FIB. All ligation junctions were sequenced to verify the identity    and orientation of the insert.

In that the structure described for naturally occurring antisense RNAsis highly similar to that for small nuclear RNAs (snRNAs), essentialcomponents of the spliceosome complex that are abundant and stable inthe nucleus of mammalian cells, the U1 snRNA gene was selected as theframework for vector construction (Guthrie, et al., Annu. Rev. Genet.,22; 387, 1988). Other attributes include the potent and constitutivelyactive nature of the U1 snRNA promoter, the ability of the unusualtrimethylguanosine 5′ cap and Sm protein interactions to signaltransport of U1 snRNA back into the nucleus (Hamm, et al., Cell, 62;569, 1990; Fischer, et al., Science 249; 786, 1990; Fischer, et al., J.Cell Biol. 113 Mo; 705, 1991; Plessel, et al., Mol. Cell. Biol. 14,4160, 1994), and the lack of polyadenylation of mature snRNAs, a factorwhich may favorably influence transcript trafficking and localization(Zhong, et al., Proc. Natl. Acad. Sci. USA, 91; 4258, 1994). Moreover,unlike other spliceosome components, U1 snRNA is widely dispersed in thenucleoplasm (Carmo-Fonseca, et al., EMBO J, 10; 195, 1991; Matera, etal., J. Cell. Biol., 121; 715, 1993).

As an illustrative example, the antisense targeting>core=was designed tocontain sequences exactly complementary to coding nucleotides 1-15 and17-30 of fibrillin mRNA (Corson, et al., Genomics, 17; 476, 1993;Pereira, et al., Hum. Mol. Genet., 2; 961, 1993) separated by the 22 bphammerhead ribozyme loop. Dominant-negative forms of fibrillin-1 causeMarfan syndrome, an autosomal dominant systemic disorder of connectivetissue (Dietz, et al., Nature, 352; 337, 1991). The regions ofcomplementarity were predicted to align the autocatalytic structure withthe consensus sequence for ribozyme cleavage (5′-GUC-3′) within thetarget message (Cech, et al., Annu. Rev. Biochem., 55; 599, 1986; T. R.Cech, Science, 236; 1532, 1987; T. R. Cech, Ann. Rev. Biochem, 59, 543,1990; Haseloff, et al., Nature, 334; 585, 1988). This antisensetargeting sequence was substituted for the short Sm protein bindingsequence between the two hairpin loops of U1 snRNA (Guthrie, Annu. Rev.Genet., 22; 387, 1988.) The sequence of the resulting chimeric RNA(FIG. 1) was analyzed using a program that predicts RNA structure (MikeZuker=s RNA page: http://ibc.wustl.edu/˜zuker/rna/). The boundaries ofthe targeting sequence utilized in the construct were selected tomaximize preservation of the U1 snRNA stem-loops, the ribozyme secondarystructure and the accessibility of the sequence complementary to thetarget message. Expression of the chimeric RNA was under thetranscriptional control of a region of U1 snRNA 5′-flanking sequencethat has been shown to be potent and constitutively active (Zhuang, etal., Cell, 46; 827, 1986; Maugin, et al., EMBO J., 5; 987, 1986); andAsselbeyers, et al., Mol. Biol. Rep. 17; 101, 1993).

FIG. 2 shows the sequence and predicted structure of chimerictranscripts derived from pU1/FIB. The Sm protein binding site of U1snRNA (boxed) has been substituted by an antisense targeting core(flanked by dashed lines) that is complementary to the first 30 codingnucleotides of FBN1 and contains a hammerhead ribozyme sequence in itscenter. The two stem-loop structures of U1 snRNA are maintained.Cleavage of the target message (unbolded characters) is predicted tooccur immediately following the consensus sequence 5′-GUC-3′ (arrow).The 5′ cap structure of the chimeric targeting molecule may contain 1 or3 methyl (m) groups. A sequence in the chimeric molecule (5′-AAUUGG-3′,underlined) remains highly similar to the consensus for Sm proteinbinding, PuA(U)nGPu (Pu=A or G).

A human osteosarcoma (MG63) cell line was stably transfected with eithera reporter gene expression vector (pZeoSVLacZ, Invitrogen) or thechimeric construct (pU1/FIB) by long-term selection for zeomycinresistance. Human osteosarcoma (MG63) cells (Sakai, et al., J. CellBiol., 103; 2499, 1986) were grown to 60% confluency and transfectedwith either linearized pU1/FIB or a lac Z reporter gene construct(pZeoSVLacZ, Invitrogen), used as a control. The transfections wereperformed using a DOTAP liposome formulation (Boehringer Mannheim, 1mg/ml) according to manufacturer=s instructions. Cells were grown in MEMmedia (Cellgro) with 10% FCS and 250 μg/ml of zeocin (Invitrogen), andwere maintained in selection for 14 days prior to the isolation ofclonal colonies. Mono- and polyclonal colonies were established andgrown to confluency.

FIG. 3 shows an immunohistochemical analysis of MG63 cells that werestably transfected with an expression construct (pZeoSVLacZ, Invitrogen)lacking targeting sequence (panels A and C) or pU1/FIB (panels B and D).The antibodies either recognized epitopes in fibrillin-1 (A and B) orfibronectin (C and D). Analysis was performed in duplicate for each ofthree independent clonal colonies for each construct, with identicalresults. Representative fields are shown at 63× magnification.Untransfected MG63 cells showed a pattern of protein deposition that wasindistinguishable from that for cells transfected with pZeoSVLacZ.

Fibrillin-1 was strikingly absent upon immunohistochemical analysis(Eldadah et al., J. Clin. Invest., 95; 874, 1995) using eitheranti-fibrillin-1 mAb 69 (a gift from L. Y. Sakai) or ananti-fibronection mAb (Sigma) of cells harboring pU1/FIB, while cellstransfected with pZeoSVLacZ showed a pattern of protein deposition thatwas indistinguishable from untransfected controls (FIG. 3). The linesharboring pU1/FIB could not be distinguished from mock-transfected oruntransfected cells upon immunohistochemical analysis with a monoclonalantibody to fibronectin, suggesting specificity for the targetingprocess.

Antisense RNAs expressed by pU1/FIB might inhibit fibrillin-1 expressionby multiple mechanisms. Ribozyme cleavage would remove the 5′ cap fromtargeted transcripts, an event predicted to effect their rapiddegradation. Alternatively, in the absence of ribozyme cleavage,involvement of the initiating AUG codon in duplex formation would impairtranslation.

The magnetic porous glass direct mRNA purification technique was used toisolate poly(A) RNA (according to the manufacturers instructions, CPG,Inc.) and Northern blot analyses were performed. Electrophoresis of 3.5μg of mRNA was performed under denaturing conditions, as previouslydescribed (Lehrach, et al., Biochemistry, 16; 4743, 1977; D. A.Goldberg, Proc. Natl. Acad. Sci. USA, 77; 5794, 1980). The gel wasexposed to 60 mjoules UV light to facilitate transfer of large mRNAspecies. RNA was transferred to nylon membrane using the turboblottingsystem according to manufacturer=s instructions (Schleicher & Schuell).The membrane was washed briefly in 2×SSC, crosslinked with 125 mjoulesof UV light and prehybridized in Expresshyb (Clontech) for {fraction(1/2)} hour at 68E C. Human cDNA probes encoding fibrillin-1 (nt370-1183), β-actin (Clontech), and G3PDH (Clontech) were labeled byrandom priming (Feinberg, et al., Anal. Biochem., 132; 6, 1983). Themembrane was hybridized for 1 hour at 68E C, washed first in 2×SSC with0.05% SDS at room temperature and then in 0.1×SSC with 0.1% SDS at 50EC.

FIG. 4 shows a Northern blot analysis of 3.5 μg of poly(a) RNA extractedfrom confluent MG63 cells that were untransfected (lane 1), or stablytransfected with either pZeoSVLacZ (Lane 2) or pU1/FIB (lane 3-5). Cellsfor lanes 1 and 3 were polyclonal, while cells for lanes 2, 4 and 5 werederived from monoclonal colonies. Hybridization was performed withradiolabeled cDNA probes complementary to fibrillin-1, P-actin, andG3PDH transcripts.

Northern blot analysis of multiple clonal colonies harboring pU1/FIBrevealed undetectable levels of FBN1 message (FIG. 4). FBN1 transcriptswere easily detected upon Northern analysis of RNA extracted fromuntransfected and mock-transfected cells. All cell types showedcomparable amounts of P-actin and G3PDH RNA, confirming the specificityof targeting.

A similar U1 construct was created containing U1 snRNA stemloopstructures and an antisense core aimed at inhibiting rent-1 expression.Functional analysis of transfected cells showed a significantup-regulation of the steady-state abundance of transcripts derived fromendogenous nonsense alleles.

SUMMARY

These data suggest that most, if not all of the inhibitory effect wasachieved at the level of target message abundance, presumably byreducing mRNA stability. Although an influence upon transcriptionalefficiency cannot be excluded, it is not intuitive given the site andnature of the predicted targeting sequence-target interaction. Basedupon previous studies, the antisense-induced mRNA degradation mostlikely takes place within the nucleus (Murray, et al., in Modern CellBiology, J. A. H. Murray, Ed. (Wiley/Liss, New York, 1992), pp 1-49 M.Cornelissen, Nucleic Acids Res., 17; 7203, 1989.). Three elements ofnuclear pre-U1 RNA, the 5′ m7G cap, the 3′ terminal stem loop, andsequences in the 5′-terminal 124 nucleotides, contribute to thestability of pre-U1 and snRNA and to efficient nuclear export (Neuman deVegvar, et al., Mol. Cell. Biol., 10; 3365, 1990; Terns, et al., GenesDev., 7; 1898, 1993; Yuo, et al., ibid., 3; 697, 1989; Izaurralde, etal., Nature, 376; 709, 1995). Nuclear targeting of cytoplasmic U1 snRNAis influenced by hypermethylation of the 5′ cap structure and binding ofat least one common U snRNP protein, both dependent upon the integrityof the Sm protein binding site which has been altered in pU1/FIB(Guthrie, et al., Annu. Rev. Genet., 22; 387, 1988). A sequence at the5′ end of the insert (AAUUGG) is highly similar to the consensus site[PuA(U)nGPu, Pu=A or G] for Sm protein binding (Jones, et al., EMBO J.,9; 2555, 1990.). It has also been shown that Sm binding sites are highlytolerant of mutations including internal nucleotide substitutions anddeletions (Jones, et al., supra).

Stable transfectin of cultured cells with an expression vectorcontaining the isolated FBN1/ribozyme core sequence of pU1/FIB but no U1snRNA secondary structure, under the transcriptional control of the SV40promoter, resulted in no discernible inhibitory effect on the expressionof fibrillin-1 transcript or protein. The oligonucleotides describedabove containing the 24 highly conserved nucleotides of hammerheadribozymes flanked by complementary sequence for 10 codons in thetranslation start site region of the FBN1 mRNA, were cloned into theSpeI and EcORI restriction sites for the polylinker of pZeoSV(Invitrogen). Fetal fibroblasts were stably transfected with thisvector. Immunohistochemical analysis of these clones was performed bymethods identical to those described herein). These results suggest thatselected properties of U1 snRNA and/or its promoter are contributing tothe extreme efficiency of inhibition seen with the use of the chimericcRNA.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A nucleic acid construct for suppressing gene expression comprisingin 5′ to 3′ operable orientation: a 5′ stem loop structure; an antisensenucleic acid; and a 3′ stem loop structure wherein the antisense nucleicacid suppresses gene expression and is flanked by the stem loopstructures and with the proviso that the antisense nucleic acid is notwithin a stem loop structure of the construct.
 2. The nucleic acidconstruct of claim 1, wherein the stem loop structures are unmodified UsnRNA structures.
 3. The nucleic acid construct of claim 2, wherein theU snRNA is U1.
 4. The nucleic acid construct of claim 1, furthercomprising a promoter.
 5. The nucleic acid construct of claim 4, whereinthe promoter is a U1 snRNA promoter.
 6. The nucleic acid construct ofclaim 4, wherein the promoter is a constitutive promoter.
 7. The nucleicacid construct of claim 4, wherein the promoter is an induciblepromoter.
 8. The nucleic acid construct of claim 1, further comprising aribozyme nucleic acid.
 9. The nucleic acid construct of claim 8, whereinthe ribozyme nucleic acid is located between the 5′ and 3′ stem loopstructures.
 10. The nucleic acid construct of claim 8, wherein theribozyme nucleic acid is a hammerhead-type ribozyme.
 11. The nucleicacid construct of claim 8, wherein a consensus sequence for ribozymecleavage in a target nucleic acid is 5′-GUC-3′ or 5′-GUA-3′.
 12. Thenucleic acid construct of claim 1, wherein the antisense nucleic acid isselected from the group consisting of rent-1, HPV E6, HIV, hyaluronicacid synthase, and fibrillin.
 13. A method for suppression of geneexpression comprising administering to a cell in vitro asuppressive-effective amount of the nucleic acid construct of claim 1,whereby expression of the gene is suppressed in the cell. 14-19(Canceled)